Integrins are α/β heterodimers and act as cell surface receptors that mediate cell to cell, or cell to extracellular matrix adhesion. Integrins connect diverse extracellular ligands to the cytoskeleton and regulate cell growth and differentiation. Hynes (2002) Cell 110: 673. The primary function of most of the twenty-four vertebrate integrins is to mediate cell adhesion and migration; in contrast, integrins αvβ6 and βvβ8 are specialized to activate TGF-β1 and β3. Munger et al. (1999) Cell 96: 319; and Mu et al. (2002) J. Cell Biol. 157: 493. The similarity in phenotypes of mice deficient in TGF-β1 (Shull et al. (1992) Nature 359: 693), integrin αvβ6 (Munger et al. (1999) Cell 96: 319) and αvβ8 (Mu et al. (2002) J. Cell Biol. 157: 493), and mice in which RGE in pro-TGF-β1 replaces RGD (Yang et al. (2007) J. Cell Biol. 176: 787), shows the importance of the RGD motif and integrins αvβ6 and αvβ8 in TGF-β1 activation in vivo. How integrins αvβ6 and αvβ8 achieve specificity, and how integrin β-subunits in general contribute to ligand specificity remains unclear. Little is known beyond mutational evidence for the importance of a disulfide-bonded loop (the β2-β3 loop) in the βI domain (Takagi et al. (1997) J. Biol. Chem. 272: 19794), and invariant binding of the metal ion dependent adhesion site (MIDAS) to an acidic residue present in all integrin ligands (Xiong et al. (2002) Science 296: 151; Xiao et al. (2004) Nature 432: 59; Nagae et al. (2012) J. Cell Biol. 197: 131; and Sen et al. (2013) J. Cell Biol. 203: 629). The issue of how the β-subunit contributes specificity is particularly acute for the five RGD-recognizing integrins that contain the acv subunit and only differ in having the β1, β3, β5, β6, or β8 subunit.
Further, integrins represent a target for treatment of various diseases or disorders, including, e.g., inflammatory diseases, anti-angiogenic therapy, and anti-thrombotic therapy, among others. Thus, screening for and identifying new small molecules that bind to the integrin ligand binding site and block interaction with its natural ligand can facilitate drug discovery process.
However, the fact that functional integrins are dimeric molecules makes study and screening of molecules that affect their function and interactions challenging. Therefore, there remains a need for methods to facilitate methods and assays for screening and characterizing integrin-ligand interaction, and thus identifying integrin ligands, e.g., for therapeutic treatment.